Luminescent Lanthanide Binding Chelates

ABSTRACT

Lanthanide chelates derived from diazacrown ethers having two ethyliminodiacetic acid side chains have increased ability to bind lanthanide ions.

This work was supported by Federal Grant Nos. NIH AR44420 and NSF9984841. The U.S. government may have rights in any patent issuing onthis application.

FIELD OF THE INVENTION

The field of the invention is luminescent lanthanide binding chelates.

BACKGROUND OF THE INVENTION

Luminescence Resonance Energy Transfer (LRET) is a modification andimprovement on the widely used technique of fluorescence resonanceenergy transfer (FRET), and can be widely used in accurately determiningthe distances between two sites bearing energy donor and energy acceptorrespectively in a bio-molecule [1]. In LRET, one of the energy donors isa luminescent lanthanide atom enhanced by a small chelate (1):

and the acceptor is a conventional (organic) fluorophore.

LRET has great distance accuracy and range; ability to resolve multipleD-A distances; great ability to isolate signal from proteins labeledwith both donor and acceptor, even in the presence of proteins labeledonly with donor or only with acceptor; and less sensitivity of energytransfer to orientation of dyes (which is often unknown).

The fundamental advantages of LRET arise because the donor emission islong-lived with millisecond lifetime compared to nanosecond lifetime ofacceptor or conventional dyes, is sharply-spiked (peaks of a fewnanometer width), has a high quantum yield [2], and is unpolarized [3].Also, the chelate's atomic structure has also been determined [4].

An order of magnitude greater accuracy in distance-determination isachieved with LRET because the energy transfer process is dominated bythe distance between the donor and acceptor, and their relativeorientations play only a minor role in determining energy transferefficiency. (A worst case scenario is 12% uncertainty in distancedetermination due to orientation effect.) This advantage is becauseterbium donor emission is unpolarized [3]. This contrasts to FRET wherethe errors due to orientation effects can be unbounded. We have shownthat angstrom changes due to protein conformational changes can readilybe measured with LRET [5, 6].

A 100-fold improvement in signal to background (S/B) is achieved withLRET. Specifically, energy transfer can be measured with essentially nocontaminating background, a stark-contrast to FRET. By temporal andspectral discrimination, donor emission and acceptor emission—bothintensity and lifetime—can be independently measured. This leads todramatically improved signal to background compared to FRET.Specifically, in LRET the acceptor emission due only to energytransfer—called sensitized emission—can be measured with no background.Contaminating background in FRET when trying to measure energy transfervia an increase in acceptor fluorescence, arises from two sources:direct excitation of the acceptor by the excitation light and donoremission at wavelengths where one looks for acceptor emission. In LRETboth sources are eliminated. For example, by choosing an acceptor suchas fluorescein and looking around 520 nm, donor emission is dark. Byusing pulsed excitation and collecting light at 520 nm only after a fewtens of microseconds, all the direct acceptor emission (which hasnanosecond lifetime) has decayed away. Samples that contain donor-onlyor acceptor-only can be spectrally and temporally discriminated againstwith LRET. Often when labeling proteins, particularly in living cells,one gets an unknown distribution of donor-donor, donor-acceptor, andacceptor-acceptor mixture. In FRET this makes distance-determinationdifficult. In LRET, sensitized emission from acceptor arises only fromdonor-acceptor labeled complex. Energy transfer of this D-A labeledcomplex can then be determined by comparing the lifetime of sensitizedemission (τ_(ad)), which decays with micro- to millisecond lifetime ofdonor that is transferring energy to the acceptor, with the donor-onlylifetime (τ_(d)): E=1−τ_(ad)/τ_(d). This ability to measure energytransfer even in complex labeling mixtures is essential for the LRETstudies on ion channels [5, 13].

We have published a number of papers on LRET (partially reviewed [7, 8])showing its advantage in model systems such as DNA oligomers [9, 10],the ability to measure distance changes of an angstrom reliably even onlarge protein complexes such as actomyosin [11, 12], and most recently,in ion channels in living cells [5,13]. Other workers have nowsuccessfully used the technique on DNA-protein complexes [14-17],actomyosin [18], protein-protein interactions in cells [19], anddetection of binding of many different biomolecules [20-22].

The current chelate-complex (1) works moderately well with both terbiumand europium. The disadvantage of such chelate-complexes is that eitherthe relatively low stability constant or fast dissociation andtransmetalation kinetics limits their application in physiologicalenvironment. The lanthanide complex of 1,4,7,10-tetraazacyclododecaneN,N′,N″,N′″-tetraacetic acid (DOTA) (2A) has been shown to be anexcellent lanthanide chelate with a large thermal and kinetic stabilityconstant, and has been widely used as a contrast agent in MRI imaging.Its non-reactive form of luminescent chelate, (DOTA)-cs124 (2B) has beensynthesized.

But it has its limitations as well. The binding of DOTA and lanthanideions is a kinetically slow process [23]. Furthermore, as for luminescentlanthanide probes, amine- or thiol-reactive groups facilitate attachmentto a biomolecule. However, neither amine-reactive nor thio-reactiveforms of DOTA-based fluorescent chelates have been reported.

The class of macrocycles known as crown ethers has been widely studiedsince their metal ion-coordinating capabilities were first reported byC. J. Pedersen (J. Am. Chem. Soc. 1967, 89, 7017). Derivations of thecrown ether include the replacement of one or more of the ring's oxygenatoms with nitrogen atoms resulting in azacrown ethers and/or theattachment of one or more side chains to the ring to form a so-calledlariat or armed crown ether. There are numerous publications on themetal-complexing properties of diazacrown ethers containing side chainsattached to the nitrogen atoms of the macrocycle (see e.g. Chi et al,Bull. Korean Chem. Soc. (2002) 23(5) 688-692; Gonzalez-Lorenzo et al,Inorg Chem. (2005) 44(12): 4254-4262; Wang et al., Chinese ChemicalLetters, (2003) 14(6): 579-580; Peters et al, J. Chem. Soc., DaltonTrans., (2000) 4664-4668; and I. A. Fallis, Annu. Rep. Prog. Chem. A 94(1998) 351-387).

We have synthesized a new type of lanthanide chelate derived fromdiazacrown ethers. Our chelates contain two ethyliminodiacetic acid sidechains and have increased ability to bind lanthanide ions.

SUMMARY OF THE INVENTION

One aspect of the invention is a crown ether lanthanide chelate ofFormula I:

or a dianhydride thereof wherein: the dotted line (----) represents asingle bond or [CH₂—O—CH₂]n′; R₁ and R₂ are independently selected fromOH, a photosensitizer, a linker optionally conjugated to a biomolecule,and a biomolecule; and n and n′ are independent integers; wherein one ormore oxygen and/or carbon atoms of the central ring of Formula I may beoptionally replaced by a protected nitrogen atom.

In a particular embodiment of the lanthanide chelate of Formula I, n is1 and the dotted line represents a single bond. In further embodiments,n is 1, the dotted line represents a single bond, R₁ is OH, and R₂ isOH.

In another embodiment of the lanthanide chelate of Formula I, n is 1 andthe dotted line represents CH₂—O—CH₂. In further embodiments, n is 1,the dotted line represents CH₂—O—CH₂, R₁ is OH, and R₂ is OH.

In one embodiment of the lanthanide chelate of Formula I, R₁ is aphotosensitizer selected from the group consisting of an aminoquinolone,an aminocoumarin, an aminoacetophenone, an aminobenzophenone, anaminofluorenone, an aminoxantone, an amino-azaxanthone, anaminoanthraquinone, and an aminoacridone sensitizer. In specificembodiments, the photosensitizer is selected from the group consistingof carbostyril 124 (7-amino-4-methyl-2-quinolinol), coumarin 120(7-amino-4-methyl-2-coumarin), and coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin).

In one embodiment of the lanthanide chelate of Formula I, R₂ is a linkerfor conjugation to a biomolecule. In particular embodiments, the linkeris a thiol-reactive or amine-reactive linker. In specific embodiments,the linker is selected from the group consisting of a maleimide moiety,a bromoacetamide moiety, a pyridyldithio moiety, an iodoacetamidemoiety, a methanethiosulfonate moiety, an isothiocyanate moiety, and anN-hydroxysuccinimide ester moiety.

In further embodiments of the lanthanide chelate of Formula I, R₁ is aphotosensitizer and R₂ is a biomolecule or a linker optionallyconjugated to a biomolecule.

In one embodiment, the lanthanide chelate of Formula I is complexed witha lanthanide ion selected from the group consisting of Tb³⁺, Eu³⁺, Lu³⁺,Dy³⁺, and Gd³⁺.

Another aspect of the invention is a method for determining aninteraction between biomolecules based on fluorescence resonance energytransfer, the method comprising: conjugating a lanthanide chelate ofFormula I via a linker at the R₂ position to a first biomolecule,wherein R₁ is a photosensitizer; labeling a second biomolecule with afluorescent energy acceptor; and measuring the resulting fluorescence.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

We disclose a new type of crown ether lanthanide chelate derived frompolyaza crown ethers that has increased ability to bind lanthanide ions.In a preferred embodiment, the lanthanide has the following Formula (I):

or is a dianhydride thereof (e.g. see structure 13). In Formula I thedotted line (----) represents a single bond or [CH₂—O—CH₂]n′; R₁ and R₂are independently selected from OH, a photosensitizer, a linkeroptionally conjugated to a biomolecule, and a biomolecule; and n and n′are independent (i.e. the same or different) integers. While thecentral, crown ether ring of Formula I can be of any size, lanthanidebinding capacity decreases with increased ring size. Thus, in preferredembodiments, n and n′ are each independently integers from 1 to 10,preferably from 1 to 5, and more preferably from 1 to 3. Equivalentcrown ether ring structures may have one or more oxygen or carbon atomsin the ring replaced by a nitrogen atom, resulting in a triaza,tetraaza, etc. crown ethers, provided that the additional nitrogen atomsare protected (e.g. with a methyl, ethyl, or other protecting group)such that only two of the nitrogen atoms of the ring carry thedicarboxylic acid side chains.

Exemplary configurations of the central ring of Formula I include1-oxa-4,7-diazacyclononane; 1,7-dioxa-4,10-diazacyclododecane;1,7-diaza-4,10,13-trioxacyclopentadecane; 1,7-diaza4,10,13,16-tetraoxacyclooctadecane; 1,10-diaza4,7,13,16-tetraoxacyclooctadecane, etc. In one embodiment of thelanthanide chelate of Formula I, n is 1 and the dotted line represents asingle bond (i.e. the ring is 1-oxa-4,7-diazacyclononane). In furtherembodiments, n is 1, the dotted line represents a single bond, R₁ is OH,and R₂ is OH. In another embodiment of the lanthanide chelate of FormulaI, n is 1 and the dotted line represents CH₂—O—CH₂ (i.e. the ring is1,7-dioxa, 4,10-diazacyclododecane). In further embodiments, n is 1, thedotted line represents CH₂—O—CH₂, R₁ is OH, and R₂ is OH. Synthesis ofthe 1-oxa-4,7-diazacyclononane- and 1,7-dioxa,4,10-diazacyclododecane-based lanthanide chelates of the invention isdetailed in Example 1.

In particular embodiments of the lanthanide chelate, R₁ and/or R₂ is aphotosensitizer. Suitable photosensitizers are known in the art (seee.g. U.S. Pat. Nos. 5,639,615 and 6,740,756) and include, for example,aminoquinolones, aminocoumarins, aminoacetophenones, aminobenzophenones,aminofluorenones, aminoxantones, amino-azaxanthones,aminoanthraquinones, and aminoacridones. In particular embodiments ofthe lanthanide chelate, R₁ is a photosensitizer selected from the groupconsisting of carbostyril 124 (7-amino-4-methyl-2-quinolinol), coumarin120 (7-amino-4-methyl-2-coumarin), and coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin). Synthesis of lanthanidechelates having a carbostyril 124 photosensitizer is detailed in Example1.

In particular embodiments of the lanthanide chelate, R₁ and/or R₂ is alinker for conjugation to a biomolecule. In preferred embodiments thelinker is thiol-reactive (see e.g. Ge P, Selvin P R, Bioconjug Chem.(2003) 14:870-876; Chen J, Selvin P R, Bioconjug Chem. (1999)10:311-315) or amine-reactive (see Li M, Selvin P R, Bioconjug Chem.(1997) 8:127-132). Exemplary linkers for conjugation to a biomoleculeinclude a maleimide moiety, a bromoacetamide moiety, a pyridyldithiomoiety, an iodoacetamide moiety, a methanethiosulfonate moiety, anisothiocyanate moiety, and an N-hydroxysuccinimide (NHS) ester moiety.Synthesis of a lanthanide chelate having a maleimide linker is detailedin Example 1 (see structure 17). An exemplary lanthanide chelate havingan NHS ester linker is depicted below (3).

In particular embodiments of the lanthanide chelate, R₁ and/or R₂ is abiomolecule optionally conjugated to the lanthanide chelate via alinker. Examples of biomolecules include proteins, polynucleotides,peptides, living cells, etc. In one embodiment, R₁ and R₂ are polylysinemolecules, suitable for use in MRI applications. The chelate may bedirectly conjugated to the biomolecule, for example as when usinganhydride-based conjugation with an amine- or thiol-containingbiomolecule such as an amine-modified DNA (e.g. DNA having a 5′C6-aminolinker; see Li M, Selvin P R, Bioconjug Chem. (1997) 8:127-132).

In particular embodiments the lanthanide chelate is complexed with alanthanide ion. In particular embodiments, the lanthanide ion isselected from Tb³⁺, Eu³⁺, Lu³⁺, Dy³⁺, and Gd³⁺.

The disclosed lanthanide chelates are useful in the same applications asprior lanthanide chelates (e.g. Gd-DTPA), such as FRET, LRET, MRI, etc,or as phasing agents in solving the crystal structures of biomolecules.One aspect of the invention is a method for determining an interactionbetween biomolecules based on fluorescence resonance energy transfer(FRET). The method comprises conjugating a lanthanide chelate of FormulaI via a linker at the R₂ position to a first biomolecule, wherein R₁ isa photosensitizer; labeling a second biomolecule with a fluorescentenergy acceptor; and measuring the resulting fluorescence. Thefluorescent energy acceptor can be any conventional fluorophore used inFRET assays such as tetramethylrhodamine iodoacetamide (TMRIA),fluorescein iodoacetanide (FIA), ATTO 465 maleimide, etc. The first andsecond biomolecules may be any molecule pair analyzable in FRET-basedassays, for example as in FRET-based detection of antibody/antigenbinding, enzyme/substrate reactions, receptor/ligand binding, etc.Resulting fluorescence is measured using routine methodology (seeExample 2).

Example 1 Synthesis and Characterization of Luminescent LanthanideBinding Chelates

We synthesized a new type of lanthanide chelate derived fromN,N′-disubstituted 12- or 9-membered (poly)-oxa-polyaza macrocycles(4,5).

Structurally, these new compounds are similar to that of DTPA (8), withthe key features include that they are 10- or 9-dentate chelates withfour ionizable carboxylate groups.

Yet, a significant difference of these chelates is that they havemacrocycle units incubated in the backbone, which can increase theirbinding ability to lanthanide ions. Because lanthanide ions can take upto 10 coordination atoms, the new chelates provide better protection tothe lanthanide ions from solvent molecule attacks, and thus longerlifetime in aqueous media compared to that of DTPA chelate (3), which isan 8-dentate chelate. For instance, TTHA (9) is a 10-dentate linearchelate. The Tb³⁺ lifetime of its TTHA-cs124 complex is longer than thatof DTPA-cs124 (2.10 ms vs 1.55 ms) [24]. And also because the structuresof these chelates are more open and flexible compared to that of DOTAchelates, the binding of lanthanide ions to the chelates is quicker thanthat of DOTA and lanthanide ions. In addition to forming a strongerbinding luminescent lanthanide probes, these chelates can also serve asgood MRI contrast agents by binding with Gd³⁺ [25].

Like DTPA, the free carboxylic acid form of the chelate can be easilyconverted to dianhydride form (7). The dianhydride form allowsattachment of an antenna molecule and either an amine-reactive group ora thiol-reactive group to the chelate to make a luminescent probe forLRET or FRET experiments.

The syntheses of the chelates are straightforward.1,7-dioxa-4,10-diazacyclododecane (9) or 1,4,7-octahydro-oxadiazonine(14) (both commercially available) are reacted withN,N-bis[(tert-butoxycarbonyl)methyl]-2-bromoethylamine (10) [26],followed by hydrolysis under either acidic or basic conditions to formthe free acid form of chelate (12). The free acid form of chelates canthen be converted to dianhydride form (13) by reaction with aceticanhydride (Ac₂O):

To synthesize amine or thiol-reactive lanthanide chelates, similarmethods used for synthesizing DTPA based chelates are employed. Theformed dianhydride form of chelate is consequently reacted with anantenna molecule (e.g. cs124) and a bi-functional thiol-reactive oramine-reactive compound in a one-pot reaction to form correspondingluminescent chelates. For example, to synthesize a thiol-reactiveluminescent probe, the dianhydride form of chelate (13) can react withcs 124 first in a ˜1:0.7 molar ratio, followed by reaction withβ-maleimidopropionic acid hydrazide (EMPH) (16) to form a thio-reactivemaleimide form of luminescent lanthanide probe (17):

We have synthesized the free acid form of 1-oxa-4,7-diazacyclononane and1,7-dioxa-4,10-diazacyclododecane based luminescent lanthanide chelates(4, 5). This free acid form of chelates was attached to cs 124 byreacting with isobutyl chloroformate first, followed by reacting withcs124 (18, 19).

The 1-oxa-4,7-diazacyclononane based, thio-reactive forms of chelates(20, 21) were also synthesized.

All of these chelates were characterized by mass spectroscopy and UV-visabsorption spectroscopy. Luminescent spectroscopy results are listed inTable 1. Comparing the number of water molecules coordinated tolanthanide ions in the non-reactive form of chelates, we can clearly seethe new chelates provide better protection to lanthanide ions fromsolvent molecule attack. In the case of Tb³⁺—N₂O-cs124, there ispractically no solvent molecule (0.02 on average) coordinated to themetal ion. Generally, the 1-oxa-4,7-diazacyclononane derived chelatesexhibit better photophysical properties in terms of brightness,lifetimes and no of water coordinated.

TABLE 1 Photophysics Data of Luminescent Lanthanide Binding ChelatesτH₂O/ No of Relative Metal Chelates τH₂O τD₂O τD₂ waters Brightness Tb³⁺DTPA-cs124 1.55 2.63 0.59 1.1(24) 1 N₂O-cs124 1.89 1.91 0.99 0.02 1.3N₂O₂-cs124 2.50 2.88 0.87 0.22 0.2 N₂O₂-cs124- 1.93(81%) 0.4 EMPH0.72(19%) Eu³⁺ DTPA-cs124 0.62 2.42 0.26 1.26(24) 1 N₂O-cs124 1.0 2.50.4 0.63 0.7 N₂O₂-cs124 N/A N₂O₂-cs124- 1.03(41%) 0.3 EMPH 0.60(22%)0.04(37%)

Example 2 LRET Measurements in Myosin

This experiment, which is adapted from Burmeister Getz et al. (BiophysJ. (1998) 74:2451-2458), demonstrates that LRET measurements on purifiedheavy meromyosin (HMM) are capable of measuring the requisite distancesbetween catalytic and light chain domains, that the measured distance inthe absence of nucleotide is consistent with the crystal structure, andthat myosin adopts a different conformation upon binding actin and actinplus ADP.

Acceptor labeling: A 5:1 mole ratio of 5-tetramethylrhodamineiodoacetamide (TMRIA) (Molecular Probes. Eugene, Oreg.) to purifiedheavy meromyosin (HMM) (isolated from rabbit skeletal muscle) is reactedovernight on ice in rigor buffer (1 mM EGTA, 5 mM MgCl₂, 20 mM MOPS, pH7.0). HMM concentrations during labeling are approx. 15 μM. The reactionis quenched by the addition of 10 mM dithiothreitol (DTT), and passedover a G-75 Sephadex size-exclusion column to remove free TMRIA. Thegoal is to achieve 2 TMRIA/HMM, with one TMRIA at each Cys707 site onthe HMM dimer.

Donor labeling: Donor chelate is placed on the light chain domain asfollows. A thio-reactive maleimide form of luminescent lanthanidechelate (17) is prepared as described in Example 1. A solution of TbCl₃is added at a 0.9:1 molar ratio to chelate at pH 7 at millimolarconcentration, and the metal is allowed to bind for 30 min on ice. Anapprox. 20-fold excess of the metal-containing chelate is then added tochicken gizzard regulatory light chain (RLC) in exchange buffer with 5mM tris-(2-carboxyethyl)phosphine hydrochloride (TCEP). The reaction isallowed to proceed for more than an hour (often overnight) at pH 7.0 onice and quenched with 10 mM DTT. Gizzard RLC contains a unique cysteine(Cys108, equivalent in position to Val 103 on the skeletal RLC, based onsequence alignment (Collins, J. Muscle Res. Cell Motil. (1991) 12:3-25).

Exchange reaction: Endogenous RLC is replaced with chelate-labeledgizzard RLC as follows. A 5- to 10-fold excess of Tb-gizzard RLC isadded to HMM (either unlabeled or TMRIA-labeled) in exchange buffer (1mM ADP, 50 mM KCl, 10 mM EDTA, 10 mM KH2PO4 (or 50 mM3-(N-morpholino)propanesulfonic acid, MOPS), pH 7.0), and the solutionis heated to 34° C. for 15 min, followed by cooling on ice and theaddition of TES (pH 7.0) and then MgCl₂ to final concentrations of 100mM and 15 mM (5 mM free Mg), respectively. Unincorporated RLC iseliminated, and the solvent changed, by passing over a G-75 columnequilibrated in rigor buffer. Incorporation of gizzard RLC into skeletalHMM is confirmed by SDS-PAGE.

To check for nonspecific binding of the gizzard RLC to HMM, the two aremixed at concentrations identical to those used for exchange (5-8 μMHMM, 25-80 μM gizzard RLC). This mixture is left on ice for 15 mininstead of being heated to 34° C., and then passed over a G75 column.During the heating step of the exchange reaction, 1 mM ADP is used topreserve the enzymatic activity of the HMM. In the presence of 1 mM ADP,both the K⁺-ATPase and actin-activated Mg²⁺-ATPase activities of HMM areunchanged after gizzard RLC exchange relative to untreated HMM.

Lanthanide luminescence measurements: All terbium emission data isrecorded on a laboratory-built spectrophotometer described previously[9] and upgraded to include a CCD for spectral measurements [4]. Samplesare placed in a quartz cuvette (either 3 mm×3 mm or 2 mm×2 mm innerdimensions) at room temperature. The concentration of HMM is typically 1μM in rigor buffer. The concentration of actin, when present, istypically 4-10 μM. This actin concentration ensures complete binding ofHMM. The terbium donor is excited with 400-1600 excitation pulses from anitrogen laser (337 nm, 5-ns pulsewidth, 40-Hz repetition rate), andterbium emission (546 nm) is acquired after passing through a gratingspectrometer with a photon-counting photomultiplier attached to amultichannel analyzer (2-ms resolution).

Curve-fitting and energy transfer analysis: Multiexponential fits aremade with Tablecurve (Jandel Scientific, Marin, Calif.). Donor-only dataare fit to two exponentials and show no residual structure.Donor-acceptor data are fit to three exponentials and also show noresidual structure. The efficiency of energy transfer is calculated fromthe lifetimes of donor luminescence as 1−(τ_(D/A)/t_(D)), where τ_(D)and τ_(D/A) are the donor excited state lifetimes in the absence andpresence of acceptor, respectively. For each experiment, donor-only anddonor-acceptor samples are prepared simultaneously, and all energytransfer calculations pair the donor-acceptor measurement with thecorresponding donor-only control. This pairwise method of comparisonyields highly reproducible results and is superior to determining energytransfer by comparing the average of donor-only lifetimes to the averageof donor-acceptor lifetimes. A paired sample t-test is used to determinethe statistical significance of differences in energy transfermeasurements between experimental conditions (HMM alone, HMM+actin,HMM+actin+ATP, HMM+actin+ADP).

Polarization measurements: Steady-state anisotropy measurements[(I_(∥)−I_(⊥))□(I_(∥)+2 I_(⊥))] of TMRIA bound to myosin are performedaccording to standard methods using 514-nm vertically polarizedexcitation, a rotatable analyzer, and a second analyzer placed at 45° toeliminate detection polarization effects. In addition, an aperture isplaced in the emission path to limit the numerical aperture, and a CCDis used as the detector [7]. Blank measurements on unlabeled myosin andunlabeled myosin bound to actin are subtracted from all signals.Measurements are performed at room temperature at ˜0.5 μM TMRIA in a 3mm×3 mm cuvette.

Steady-state anisotropy measurements on the terbium-labeled gizzard RLCexchanged into HMM (without TMRIA) are performed similarly, except thatexcitation is with vertically polarized 337-nm pulsed light, and theemission is passed through a single analyzer and a chopper before beingdetected by the CCD. The polarization sensitivity of the optics isdetermined by assuming that the anisotropy of a Tb-DTPA-cs 124 chelatefreely diffusing in solution is zero. (The spectrometer has a bias infavor of horizontally polarized light.)

Results: The results show that LRET measurements on HMM are capable ofmeasuring the requisite distances between catalytic and light chaindomains, that the measured distance in the absence of nucleotide isconsistent with the crystal structure, and that myosin adopts adifferent conformation upon binding actin and actin plus ADP.

The foregoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications and patentapplications cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims

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1. A crown ether lanthanide chelate of Formula I:

or a dianhydride thereof wherein: the dotted line (----) represents asingle bond or [CH₂—O—CH₂]n′; R₁ and R₂ are independently selected fromOH, a photosensitizer, a linker optionally conjugated to a biomolecule,and a biomolecule; and n and n′ are independent integers; wherein one ormore oxygen and/or carbon atoms of the central ring of Formula I may beoptionally replaced by a protected nitrogen atom.
 2. The lanthanidechelate of claim 1 wherein n is 1 and the dotted line represents asingle bond.
 3. The lanthanide chelate of claim 1 wherein n is 1, thedotted line represents a single bond, R₁ is OH, and R₂ is OH.
 4. Thelanthanide chelate of claim 1 wherein n is 1 and the dotted linerepresents CH₂—O—CH₂.
 5. The lanthanide chelate of claim 1 wherein n is1, the dotted line represents CH₂—O—CH₂, R₁ is OH, and R₂ is OH.
 6. Thelanthanide chelate of claim 1 wherein R₁ is a photosensitizer.
 7. Thelanthanide chelate of claim 1 wherein R₁ is a photosensitizer selectedfrom the group consisting of an aminoquinolone, an aminocoumarin, anaminoacetophenone, an aminobenzophenone, an aminofluorenone, anaminoxantone, an aminoazaxanthone, an aminoanthraquinone, and anaminoacridone sensitizer.
 8. The lanthanide chelate of claim 1 whereinR₁ is a photosensitizer selected from the group consisting ofcarbostyril 124 (7-amino-4-methyl-2-quinolinol), coumarin 120(7-amino-4-methyl-2-coumarin), and coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin).
 9. The lanthanide chelate ofclaim 1 wherein R₂ is a linker for conjugation to a biomolecule.
 10. Thelanthanide chelate of claim 1 wherein R₂ is a thiol-reactive linker forconjugation to a biomolecule.
 11. The lanthanide chelate of claim 1wherein R₂ is an amine-reactive linker for conjugation to a biomolecule.12. The lanthanide chelate of claim 1 wherein R₂ is a linker forconjugation to a biomolecule, wherein the linker is selected from thegroup consisting of a maleimide moiety, a bromoacetamide moiety, apyridyldithio moiety, an iodocetamide moiety, a methanethiosulfonatemoiety, an isothiocyanate moiety, and an N-hydroxysuccinimide estermoiety.
 13. The lanthanide chelate of claim 1 wherein R₁ is aphotosensitizer and R₂ is a linker optionally conjugated to abiomolecule.
 14. The lanthanide chelate of claim 1 wherein R₂ is alinker conjugated to a biomolecule.
 15. The lanthanide chelate of claim1 wherein R₂ is a biomolecule.
 16. The lanthanide chelate of claim 1that is complexed with a lanthanide ion.
 17. The lanthanide chelate ofclaim 1 that is complexed with a lanthanide ion selected from the groupconsisting of Tb³⁺, Eu³⁺, Lu³⁺, Dy³⁺, and Gd³⁺.
 18. A method fordetermining an interaction between biomolecules based on fluorescenceresonance energy transfer, the method comprising: conjugating alanthanide chelate of claim 1 via a linker at the R₂ position to a firstbiomolecule, wherein R₁ is a photosensitizer; labeling a secondbiomolecule with a fluorescent energy acceptor; and measuring theresulting fluorescence.